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Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Preconceptual sex selection is an ethically justifiable process whereby X- and Y-chromosome bearing spermatozoa are isolated prior to fertilization of the oocyte in order to generate either a male or a female offspring. Although various separation techniques are available, none can guarantee 100% accuracy. There are various physiological differences between X- and Y-chromosome bearing spermatozoa which can be used to separate these two populations of sperm. For the purpose of this study, X- and Y-chromosome bearing spermatozoa were separated based on (1) their respective abilities to remain viable when subjected to adverse environments, including extreme pH values, increased temperatures and various hydrogen peroxide (H2O2) concentrations; (2) the ability of Y-chromosome bearing spermatozoa to swim faster and/or more progressively than X-chromosome bearing spermatozoa; and (3) the X-chromosome bearing spermatozoa’s increased size and weight when compared to the Y-chromosome bearing spermatozoa. The efficacy of live and dead cell separation through (i) Magnetic Antibody Cell Separation (MACS) and (ii) a modified swim-up technique was also assessed and compared. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. Sperm motility (CASA) and viability (eosin/nigrosin) was assessed before and after each intervention. Ethical clearance for this study was granted by the Health Research Ethics Committee 1 (Ethics #: S13/04/068). The results indicated successful enrichment of X-chromosome bearing spermatozoa upon incubation in acidic media, increased temperatures, and H2O2. In contrast, Y-chromosome bearing spermatozoa were successfully enriched through a direct swim-up method as well as discontinuous gradient centrifugation. In conclusion, this study demonstrated the potential role for physiological differences between X- and Y-chromosome bearing spermatozoa in the development of preconceptual gender selection through sperm sorting. / AFRIKAANSE OPSOMMING: Prekonsepsie geslagselektering is 'n eties regverdigbare proses waardeur X- en Y- chromosoom draende spermatosoë geïsoleer word voordat bevrugting van die oösiet plaasvind, om óf 'n manlike óf 'n vroulike nageslag te genereer. Alhoewel verskeie skeidingstegnieke beskikbaar is, kan geeneen 100% akkuraatheid waarborg nie. Daar bestaan verskeie fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë wat skeiding van hierdie twee groepe spermatosoë moontlik kan maak. Vir die doel van hierdie studie is skeidingsmetodes vir die X- en Y- chromosoom draede spermatosoë gebaseer op (1) hul onderskeie vermoëns om lewensvatbaar te bly tydens blootstelling aan ‘n ongunstige milieu, insluitend ekstreme pH waardes, verhoogde temperature en verskeie waterstofperoksied (H2O2) konsentrasies; (2) die vermoë van die Y-chromosoom draende spermatosoon om vinniger en/of meer progressief as X-chromosoom draende spermatosoë te swem; en (3 ) die X-chromosoom draende spermatosoon se verhoogde grootte en gewig in vergelyking met die Y- chromosoom draende spermatosoon. Die effektiwiteit van die (i) Magnetiese Anti-liggaam Sel Skeidingstegniek (MACS) en (ii) 'n aangepaste weergawe van die op-swem tegniek om lewendige en dooie selle te skei is ook bepaal en vergelyk. Veranderinge in die geslagschromosoom verhouding van die monsters is bepaal deur dubbel-etiket fluoresensie in situ hibridisering (FISH) voor en na verwerking. Spermmotiliteit (CASA) en lewensvatbaarheid (eosien/nigrosin) is bepaal voor en na elke intervensie. Etiese goedkeuring vir hierdie studie is verleen deur die Gesondheids-Navorsingsetiekkomitee 1 (Etiese # : S13/04/068). Die resultate dui suksesvolle verryking van X-chromosoom draende spermatosoë deur inkubasie in suur media, verhoogde temperature, en H2O2. Y-chromosoom draende spermatosoë is verryk deur middel van 'n direkte op-swem metode sowel as diskontinue gradiënt sentrifugering . Ten slotte, hierdie studie toon die potensiële rol vir fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë in die ontwikkeling van prekonsepsie geslagselektering metodes deur skeiding van X- en Y-chromosoom draende sperme.

Resumo: Diante do grande impacto da fertilidade dos touros sobre as eficiências reprodutiva e produtiva em rebanhos comerciais, o presente trabalho foi realizado visando avaliar as características testiculares e seminais como critérios de seleção de touros jovens da raça Nelore. As correlações fenotípicas foram, de modo geral, baixas ou nulas (P<0,01) entre as características testiculares, seminais e de crescimento. Todas as características testiculares ou seminais deverão ser contempladas no exame andrológico, visto a independência existente entre as mesmas e a importância destas na comprovação da fertilidade de touros. Os componentes de (co)variância e os valores genéticos foram estimados pelo método da amostragem de Gibbs pelo programa GIBBS2F90, sob modelo animal, o qual incluiu como efeitos fixos, os grupos de contemporâneos e a idade do animal e como aleatório o efeito genético aditivo direto. As estimativas de herdabilidade para perímetro escrotal aos 18 meses, perímetro escrotal no andrológico, volume testicular, formato testicular, aptidão andrológica, defeitos espermáticos maiores, menores e totais foram: 0,42; 0,41; 0,23; 0,22; 0,10; 0,16; 0,04 e 0,15. As correlações genéticas entre perímetro escrotal aos 18 meses com as demais características testiculares e aptidão andrológica foram baixas a altas, de 0,20 a 0,84 e, baixas com as seminais, de -0,16 a -0,24. A circunferência escrotal como critério de seleção deverá promover ganho genético rápido por seleção individual e, por resposta correlacionada, aumento do volume testicular, mudança para formas mais esféricas dos testículos, melhoria da qualidade seminal e aumento do número de animais aptos à reprodução nas condições extensivas de criação no Brasil. / Abstract: Considering the great impact of fertility of bulls on reproductive and productive efficiency in commercial herds, the objective of this study was to evaluate the testicular and seminal traits as selection criteria of Nellore young bulls. The residual correlations between the traits were analyzed and, in general, the phenotypic associations were small (P<0.01) or null between the testicular, seminal and growth traits. The testicular or seminal traits should be considered in breeding soundness examination, due to their independence of each other and the importance to fertility of bulls. The genetic (co)variance components and genetic values were estimated by the Gibbs sampling method, using the program GIBBS2F90, under an animal model, which included, as fixed effects, the contemporary groups and animal age and, as random effect, the additive genetic effect. The heritability estimates for scrotal circumference at 18 months, scrotal circumference, testicular volume, testicular format, breeding soundness classification, major sperm defects, minor and total sperm defects were: 0.42, 0.41, 0.23, 0.22, 0.10., 0.16, 0.04 and 0.15. Genetic correlations between scrotal circumference at 18 months and others testicular traits and breeding soundness classification were low to high (0.20 to 0.84) and, with seminal traits was low (-0.16 to - 0.24). The scrotal circumference as a selection criterion should result in rapid genetic gain by individual selection, and as correlated response, should result in testicular volume, changes to more spherical shapes of the testicles, improved sperm quality and increased number of satisfactory bulls for breeding under extensive conditions in Brazil. / Orientadora: Lúcia Galvão de Albuquerque / Coorientador: Joanir Pereira Eler / Banca: José Bento Sterman Ferraz / Banca: José Domingos Guimarães / Banca: Humberto Tonhati / Banca: Maurício Mello de Alencar / Doutor

Further in vivo functional studies were also performed. The SLC26A3 antibody was injected into the BALB/C mice seminiferous tubules using micropipette. The animals were sacrificed after three days, and CASA, daily sperm production (DSP) were used to evaluate sperm motility and spermatogenesis. The results showed that sperm motility was increased while there was no significant difference between DSP. Our results indicate that SLC26A3 on sperm does not play a dominant role in spermatogenesis, epididymal maturation and sperm motility. / In the first part of study, guinea pig sperm which were incubated in medium with various concentrations of Cl- resulted in varied percentages of capacitated sperm, in a concentration dependent manner. Depleting Cl-, even in the presence of HCO3 -, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO 3- dependent events, including cAMP production, protein tyrosine phosphorylation and pHi increase also depend on Cl - concentrations. Similar Cl- dependence was observed for sperm hyperactivated motility and sperm-egg fusion. The capacitation-associated events could also be significantly reduced by inhibitors or antibodies of CFTR and SLC26A3, with a more potent effect observed for niflumate, an inhibitor more selective for SLC26A3, over that of DIDS, an inhibitor more selective for SLC4 exchangers. The expression and localization of CFTR and SLC26A3 in guinea pig sperm were also demonstrated using immunostaining and Western blot analysis. Our results indicate that Cl- is required for the entry of HCO3- necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3 - with CFTR providing the recycling pathway for Cl- . / In the second part of study, GC-1 spg cell line that expresses SLC26A6 but not SLC26A3 was used as a negative control. The cells and sperm were pretreated with anion exchanger inhibitors and SLC26A3 antibody, and then membrane potential and intracellular calcium were measured. Our results showed that DIDS could inhibit the HCO3- deficiency induced depolarization of GC-1 spg cells as well as the depolarization induced by Cl- or HCO3- deficiency in sperm. Niflumate could inhibit the HCO3- induced [Ca 2+] i increase of the sperm but not GC-1 spg cells. SLC26A3 antibody had no effect on the GC-1 spg cells but it could block the depolarization caused by C--deficiency in sperm. / Our previous study has demonstrated the involvement of Cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate necessary for sperm capacitation. However, whether its involvement is direct or indirect remains unclear. The present study is design to investigate: (1) the possibility of a Cl-/HCO3- exchanger, solute carrier family 26, number 3 (SLC26A3), operating with CFTR during sperm capacitation, (2) the role and the underlying mechanisms of SLC26A3 in other sperm post-testicular processes and spermatogenesis. / Taken together, our results demonstrate the involvement of SLC26A3 in sperm function, particularly in transporting HCO3- necessary for sperm capacitation, which appears to be working with CFTR providing the recycling pathway for Cl- in parallel. The present results also provide an explanation to the observed subfertility in patients with SLC26A3 mutations. Further in vitro and in vivo studies also have shown that SLC26A3 does not play a predominant role in spermatogenesis but may affect other post-testicular maturation processes. / Chen, Wenying. / "November 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les biotechnologies de la reproduction sont aujourd’hui largement utilisées dans le contrôle de la fertilité animale et humaine. Ces techniques présentent cependant des rendements faibles et font actuellement l’objet de nombreuses recherches. Ce travail de thèse s’inscrit dans ce contexte et se focalise sur la recherche de nouvelles molécules pro-fertilité dans l’espèce bovine, et s’articule autour de deux axes : le premier consiste à tester les propriétés pro-fertilité d’une enzyme du métabolisme lipidique sur la maturation ovocytaire, la fécondation et le développement embryonnaire préimplantatoire in vitro, et le deuxième à découvrir des molécules permettant d’améliorer la fécondance des spermatozoïdes. Nous démontrons ici que l’application de l’enzyme améliore de manière significative le nombre et la qualité des embryons au stade blastocyste. Un savoir-faire quant à l’utilisation de cette enzyme (fenêtre de traitement, concentration,…) a été développé. Cette thèse a également permit de caractériser différents composés avec des propriétés différentes dont une molécule originale permettant d’augmenter la vitesse des spermatozoïdes. Ce composé est prometteur car il est aussi actif sur des spermatozoïdes issus du testicule, de l’épididyme ou de l’éjaculat, avant ou après congélation. / The reproductive biotechnologies are now widely used in control of animal and human fertility. However, these technics have low yields and are currently the subject of much research. In this context, the present thesis focuses on the search for new pro-fertility molecules in cattle, organized around two axis : the first one is to test the pro-fertility properties of an enzyme from lipid metabolism on maturation, fertilization and preimplantation embryo development in vitro, and the second one is to discover molecules to improve sperm fertilizing ability. Here, we show that application of the enzyme significantly improve the number and quality of embryos at the blastocyst stage. Expertise in the use of this enzyme (time of treatment, concentration, etc.) was developed. This thesis also allowed the characterization of different compounds with different properties. Among them, one original molecule increase sperm velocity. This compound is promising because it works on sperm from the testis, epididymis or ejaculate before or after freezing.

A aplicação de novos sistemas para medir as características de deslocamento espermático e sua associação com a fertilidade vem apresentando um crescimento recente. Computer-assisted semen analysis (CASA) permite uma avaliação padronizada das características da motilidade espermática e é plausível que a combinação destes parâmetros com as características morfológicas seminais possam estimar a fertilidade do ejaculado. O desempenho reprodutivo (taxa de parto - TP e número de leitões nascidos totais - NT) de 14 reprodutores suínos foi obtido a partir de um experimento conduzido em uma granja comercial com fêmeas multíparas utilizando inseminação homospérmica com deposição intra-cervical e dois bilhões de espermatozoides móveis por dose inseminante. Durante 10 semanas, as fêmeas (n=948) foram inseminadas e igualmente distribuídas entre os machos testados de acordo com a ordem de parto, média do histórico de leitões nascidos, escore de condição corporal, intervalo desmame-estro (IDE), duração da lactação e número de leitões desmamados. Características da motilidade espermática, avaliadas pelo sistema CASA in natura e durante armazenamento a 17°C por 240h, e os defeitos morfológicos avaliados no sêmen in natura, foram incluídas na análise para determinar quais parâmetros espermáticos foram importantes para discriminar o potencial reprodutivo dos ejaculados desses machos. Os dados foram submetidos à análise multivariada que incluiu procedimentos estatísticos tais como: análises de cluster, canônica e discriminante. Dentre os 14 machos avaliados, 4 clusters foram formados com base nos resultados reprodutivos. Um macho, com menor resultado de TP e NT (89,7% e 11,98) e dois machos, com os maiores valores de TP e NT (97,8% e 14,16) foram alocados em diferentes grupos. Os demais machos foram separados em outros dois grupos distintos (4 e 7 machos), incluindo machos com valores semelhantes de NT (12,64 e 13,22) mas divergentes na TP (95,9% vs. 91,8%). A análise canônica permitiu separar os grupos, sendo a melhor separação observada entre o grupo menos fértil e os demais. As características seminais que demonstraram melhor capacidade de discriminação dos grupos incluíram motilidade espermática total, motilidade progressiva, amplitude do deslocamento lateral da cabeça espermática, além das características morfológicas tal como a presença de gota citoplasmática. A análise multivariada discriminante permitiu classificar corretamente mais de 80% dos 140 ejaculados avaliados em seus respectivos grupos. A análise multivariada pode ser uma eficiente ferramenta estatística na discriminação do potencial reprodutivo de cachaços. / The application of new approaches that measure certain sperm motion characteristics and their association with boar fertility has shown a recent increase. Computer-assisted semen analysis (CASA) offers standardized assessment of multiple sperm motion parameters and it is plausible that the combination of these parameters with morphological sperm defects may indicate the fertility potential of boars. The fertility outcome (farrowing rate - FR and number of total piglets born - TB) of 14 boars was obtained from a field trial conducted in a commercial farm using multiparous sows through single sire mating with two billion motile sperm cells per AI dose. Sows (n=948) bred were equally distributed among boars according to parity, mean lifetime number of piglets born, body condition score, weaning-to-estrus interval, lactation length and number of weaned piglets during 10 weeks of breeding period. Sperm motion parameters evaluated with CASA system in natura and in semen stored at 17°C for 240h in addition to morphological sperm defects, measured at the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate statistical analysis, including cluster, canonical and discriminant analyses. Among the 14 evaluated boars four clusters were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The others boars were separated in two new distinct clusters (4 and 7 boars), including boars with intermediate TB (12.64 and 13.22) but divergent values of FR (95.9% vs. 91.8%). Canonical analysis was able to separate the individual ejaculates into the previously clustered boars and a better separation was shown between the lowest fertility group and the others. Semen traits which had higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement, analyzed during semen storage, and sperm morphological defects such as cytoplasmatic droplets. The multivariate discriminant analysis showed that more than 80% of the 140 ejaculates were correctly classified into their own group. The multivariate analysis can be an efficient statistical tool to improve the discrimination of potential fertility of boars.

Highly profitable black caviar market and the depletion of wild sturgeon stocks warrant improvements in sturgeon aquaculture. Therefore, chromosomal manipulations, particularly gynogenesis, are focused on for increasing the ratio of females over males in progeny. The present study focused on optimizing chromosomal manipulations in sturgeons, particularly gynogenesis. The reasons of low survival rates were analyzed and the critical steps of gynogenesis induction processes were optimized. In addition, alternative ways of DNA inactivation in sperms were investigated, as well as the influence of native light-dependent DNA repair mechanisms on gynogenesis induction. Methods of interspecific gynogenesis usage for simplifying gynogenetic progeny separation were also proposed. Spectrophotometry analysis was used to investigate the ability of UV light, as the most common DNA inactivating agent, to penetrate into sperm. In addition, investigation of UV-irradiated sperm motility and results of partial gynogenesis induction showed that low transparency of sperms for UV-light can cause significant heterogeneity of UV-irradiation. As a result, a proper dilution of sperm was suggested as a critical step for homogeneous UV-irradiation of samples. Gynogenesis in sterlet was induced with chemical agents that damage sperm DNA, as an alternative to UV irradiation for applied in large-scale production of gynogenotes. All tested substances showed ability to inactivate DNA in spermatozoa, and thus producing gynogenotes. Negative impact of treatments with chemical agents on the sperm motility was observed. Subsequently, these treatments had a low efficiency of gynogenesis induction. The highest percentage of produced gynogenetic larvae 19.8 ? 8.9% was obtained by treatment with aminomethyl-4,5?,8-trimethylpsoralen (AMT) at 50 ?M followed by UV-A (360 nm) irradiation at dose of 900 J/m2. Therefore, this treatment could be used as a substitute for commonly used UV-C irradiation, e.g., in the case of large volumes of sperm. Detailed investigation of photoreactivation in sturgeon sperm revealed a significant level of light-dependent DNA restoration in sperms irradiated with high doses of UV-C light. Induction of gynogenesis with UV-C irradiation followed by exposure to visible light resulted in significant deviations from the typical Hertwig effect. In contrast, the red light with a wavelength of more than 600 nm did not result in decreased DNA damage, instead a moderate increase in damage was observed, i.e., it did not induce photoreactivation. Therefore, the use of infrared light to illuminate work stations during the induction of gynogenesis is suggested. The use of interspecific gynogenesis, particularly gametes of sturgeon species with different ploidy levels, was suggested as a way to simplify the separation of gynogenotes. In addition, application of this method allowed studying the effectiveness of DNA-inactivation and ploidy restoration treatments separately, as well as evaluation of fitness parameters and survival rates in each group of progeny without the physical separation of fish. Finally, the protocol for tetraploidization in sterlet was optimized for the prospective using tetraploid individuals for the induction of gynogenesis and androgenesis with diploid eggs and sperm. In conclusion, the described methods and protocols allowed gynogenesis induction in sturgeons with a survival rate sufficient for aquaculture, taking into consideration their high fertility, although further studies of the consequences of this treatment on fish is required.

A aplicação de novos sistemas para medir as características de deslocamento espermático e sua associação com a fertilidade vem apresentando um crescimento recente. Computer-assisted semen analysis (CASA) permite uma avaliação padronizada das características da motilidade espermática e é plausível que a combinação destes parâmetros com as características morfológicas seminais possam estimar a fertilidade do ejaculado. O desempenho reprodutivo (taxa de parto - TP e número de leitões nascidos totais - NT) de 14 reprodutores suínos foi obtido a partir de um experimento conduzido em uma granja comercial com fêmeas multíparas utilizando inseminação homospérmica com deposição intra-cervical e dois bilhões de espermatozoides móveis por dose inseminante. Durante 10 semanas, as fêmeas (n=948) foram inseminadas e igualmente distribuídas entre os machos testados de acordo com a ordem de parto, média do histórico de leitões nascidos, escore de condição corporal, intervalo desmame-estro (IDE), duração da lactação e número de leitões desmamados. Características da motilidade espermática, avaliadas pelo sistema CASA in natura e durante armazenamento a 17°C por 240h, e os defeitos morfológicos avaliados no sêmen in natura, foram incluídas na análise para determinar quais parâmetros espermáticos foram importantes para discriminar o potencial reprodutivo dos ejaculados desses machos. Os dados foram submetidos à análise multivariada que incluiu procedimentos estatísticos tais como: análises de cluster, canônica e discriminante. Dentre os 14 machos avaliados, 4 clusters foram formados com base nos resultados reprodutivos. Um macho, com menor resultado de TP e NT (89,7% e 11,98) e dois machos, com os maiores valores de TP e NT (97,8% e 14,16) foram alocados em diferentes grupos. Os demais machos foram separados em outros dois grupos distintos (4 e 7 machos), incluindo machos com valores semelhantes de NT (12,64 e 13,22) mas divergentes na TP (95,9% vs. 91,8%). A análise canônica permitiu separar os grupos, sendo a melhor separação observada entre o grupo menos fértil e os demais. As características seminais que demonstraram melhor capacidade de discriminação dos grupos incluíram motilidade espermática total, motilidade progressiva, amplitude do deslocamento lateral da cabeça espermática, além das características morfológicas tal como a presença de gota citoplasmática. A análise multivariada discriminante permitiu classificar corretamente mais de 80% dos 140 ejaculados avaliados em seus respectivos grupos. A análise multivariada pode ser uma eficiente ferramenta estatística na discriminação do potencial reprodutivo de cachaços. / The application of new approaches that measure certain sperm motion characteristics and their association with boar fertility has shown a recent increase. Computer-assisted semen analysis (CASA) offers standardized assessment of multiple sperm motion parameters and it is plausible that the combination of these parameters with morphological sperm defects may indicate the fertility potential of boars. The fertility outcome (farrowing rate - FR and number of total piglets born - TB) of 14 boars was obtained from a field trial conducted in a commercial farm using multiparous sows through single sire mating with two billion motile sperm cells per AI dose. Sows (n=948) bred were equally distributed among boars according to parity, mean lifetime number of piglets born, body condition score, weaning-to-estrus interval, lactation length and number of weaned piglets during 10 weeks of breeding period. Sperm motion parameters evaluated with CASA system in natura and in semen stored at 17°C for 240h in addition to morphological sperm defects, measured at the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate statistical analysis, including cluster, canonical and discriminant analyses. Among the 14 evaluated boars four clusters were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The others boars were separated in two new distinct clusters (4 and 7 boars), including boars with intermediate TB (12.64 and 13.22) but divergent values of FR (95.9% vs. 91.8%). Canonical analysis was able to separate the individual ejaculates into the previously clustered boars and a better separation was shown between the lowest fertility group and the others. Semen traits which had higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement, analyzed during semen storage, and sperm morphological defects such as cytoplasmatic droplets. The multivariate discriminant analysis showed that more than 80% of the 140 ejaculates were correctly classified into their own group. The multivariate analysis can be an efficient statistical tool to improve the discrimination of potential fertility of boars.

A aplicação de novos sistemas para medir as características de deslocamento espermático e sua associação com a fertilidade vem apresentando um crescimento recente. Computer-assisted semen analysis (CASA) permite uma avaliação padronizada das características da motilidade espermática e é plausível que a combinação destes parâmetros com as características morfológicas seminais possam estimar a fertilidade do ejaculado. O desempenho reprodutivo (taxa de parto - TP e número de leitões nascidos totais - NT) de 14 reprodutores suínos foi obtido a partir de um experimento conduzido em uma granja comercial com fêmeas multíparas utilizando inseminação homospérmica com deposição intra-cervical e dois bilhões de espermatozoides móveis por dose inseminante. Durante 10 semanas, as fêmeas (n=948) foram inseminadas e igualmente distribuídas entre os machos testados de acordo com a ordem de parto, média do histórico de leitões nascidos, escore de condição corporal, intervalo desmame-estro (IDE), duração da lactação e número de leitões desmamados. Características da motilidade espermática, avaliadas pelo sistema CASA in natura e durante armazenamento a 17°C por 240h, e os defeitos morfológicos avaliados no sêmen in natura, foram incluídas na análise para determinar quais parâmetros espermáticos foram importantes para discriminar o potencial reprodutivo dos ejaculados desses machos. Os dados foram submetidos à análise multivariada que incluiu procedimentos estatísticos tais como: análises de cluster, canônica e discriminante. Dentre os 14 machos avaliados, 4 clusters foram formados com base nos resultados reprodutivos. Um macho, com menor resultado de TP e NT (89,7% e 11,98) e dois machos, com os maiores valores de TP e NT (97,8% e 14,16) foram alocados em diferentes grupos. Os demais machos foram separados em outros dois grupos distintos (4 e 7 machos), incluindo machos com valores semelhantes de NT (12,64 e 13,22) mas divergentes na TP (95,9% vs. 91,8%). A análise canônica permitiu separar os grupos, sendo a melhor separação observada entre o grupo menos fértil e os demais. As características seminais que demonstraram melhor capacidade de discriminação dos grupos incluíram motilidade espermática total, motilidade progressiva, amplitude do deslocamento lateral da cabeça espermática, além das características morfológicas tal como a presença de gota citoplasmática. A análise multivariada discriminante permitiu classificar corretamente mais de 80% dos 140 ejaculados avaliados em seus respectivos grupos. A análise multivariada pode ser uma eficiente ferramenta estatística na discriminação do potencial reprodutivo de cachaços. / The application of new approaches that measure certain sperm motion characteristics and their association with boar fertility has shown a recent increase. Computer-assisted semen analysis (CASA) offers standardized assessment of multiple sperm motion parameters and it is plausible that the combination of these parameters with morphological sperm defects may indicate the fertility potential of boars. The fertility outcome (farrowing rate - FR and number of total piglets born - TB) of 14 boars was obtained from a field trial conducted in a commercial farm using multiparous sows through single sire mating with two billion motile sperm cells per AI dose. Sows (n=948) bred were equally distributed among boars according to parity, mean lifetime number of piglets born, body condition score, weaning-to-estrus interval, lactation length and number of weaned piglets during 10 weeks of breeding period. Sperm motion parameters evaluated with CASA system in natura and in semen stored at 17°C for 240h in addition to morphological sperm defects, measured at the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate statistical analysis, including cluster, canonical and discriminant analyses. Among the 14 evaluated boars four clusters were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The others boars were separated in two new distinct clusters (4 and 7 boars), including boars with intermediate TB (12.64 and 13.22) but divergent values of FR (95.9% vs. 91.8%). Canonical analysis was able to separate the individual ejaculates into the previously clustered boars and a better separation was shown between the lowest fertility group and the others. Semen traits which had higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement, analyzed during semen storage, and sperm morphological defects such as cytoplasmatic droplets. The multivariate discriminant analysis showed that more than 80% of the 140 ejaculates were correctly classified into their own group. The multivariate analysis can be an efficient statistical tool to improve the discrimination of potential fertility of boars.

The motility of male gametes (sperm) is one of the important factors influencing the reproductive success of males. Because sperms are often subjected to strong postmating sexual selection and even closely related species often differ in sperm morphology, sperm motility could also differ between species, which may contribute to reproductive isolation between species. As part of my diploma thesis, I studied sperm motility in two closely related species of songbirds, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia). These two species of nightingales are an ideal model system because the areas of these two species overlap in the secondary contact zone across Central and Eastern Europe, where they occasionally hybridize and thus allow the study of speciation mechanisms in the natural environment. Both species also differ greatly in total sperm length. As part of my diploma thesis, I studied the possible influence of different sperm morphology on their motility. I further tested whether the motility of nightingale sperm differs in the fluid from the cloaca of a female of the same species and a different species, which would demonstrate the presence of postmating prezygotic reproductive isolation between species. The results of my work showed that despite the...